Proinflammatory stress activates neutral sphingomyelinase 2 based generation of a ceramide-enriched β-cell EV subpopulation

β-cell extracellular vesicles (EVs) play a role as paracrine effectors in islet health, yet mechanisms connecting β-cell stress to changes in EV cargo and potential impacts on diabetes remain poorly defined. We hypothesized that β-cell inflammatory stress engages neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways, generating ceramide-enriched small EVs that could impact surrounding β cells. Consistent with this, proinflammatory cytokine treatment of INS-1 β cells and human islets concurrently increased β-cell nSMase2 and ceramide abundance, as well as small EV ceramide species. Direct chemical activation or genetic knockdown of nSMase2, chemical treatment to inhibit cell death pathways, or treatment with a GLP-1 receptor agonist also modulated β-cell EV ceramide. RNA sequencing of ceramide-enriched EVs identified a distinct set of miRNAs linked to β-cell function and identity. EV treatment from cytokine-exposed parent cells inhibited peak GSIS in wild-type recipient cells; this effect was abrogated when using EVs from nSMase2 knockdown parent cells. Finally, plasma EVs in children with recent-onset T1D showed increases in multiple ceramide species. These findings highlight nSMase2 as a regulator of β-cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of β-cell inflammatory stress and death.